90% of the soil on the earth is inadequate soil that has some kind of problem. The inadequate soil, in general, lacks the elements essential to the growth of plants qualitatively and quantitatively, and therefore, the growth of plants is hindered or growth disorders occur due to the soil containing a large amount of heavy metals. Representative of inadequate soil is dryland salt accumulated soil. Of this type, there are ones in which NaCl and Na2CO3 are accumulated or CaCO3 or CaSO4 are accumulated in the topsoil due to artificial over-irrigation or dry weather over a long period of time. The halomorphic soil causes a salt density disorder, and the calcareous soil causes an iron deficiency disorder.
Approximately 30% of the cultivated soil on the earth is said to be a potentially iron deficient area. (Wallence et al. “Iron Chlorosis in Horticultural Plants,” 75 American Society for Horticultural Science, 819–839 (1960)). The calcareous soil in semiarid areas has calcareous components eluted from the core material due to a capillary effect and it is accumulated on the surface of the ground. In this soil, the pH is increased and becomes alkaline, and therefore, the iron in the soil exists in the form of Fe (OH)3 and has extremely low solubility.
The plants grown in these soils have iron deficient chlorosis due to little soluble iron, and their growth is hindered or they die.
The iron obtaining system of higher plants is classified into two types, Strategy I and Strategy II. Strategy I is an iron obtaining system for higher plants excluding gramineae. It is a system in which the insoluble trivalent iron in the soil is reduced by the trivalent iron reduction enzyme that is present on the surface of the cell on the root, and then is it absorbed by the divalent iron transporter. Of those plants that have this system, there are ones that have a system to emit protons in the rhizosphere to increase the activity of the trivalent iron reduction enzyme by lowering the pH in the rhizosphere, and ones that have a system to emit phenol compounds in the rhizosphere and supply the Fe (III) to the trivalent reduction enzyme that is present on the surface of the cell by an Fe (III)-phenol compound chelate. Recent studies have isolated the divalent iron transporter IRT1 (Eide et al., “A novel iron-regulated metal transporter from plants identified by functional expression in yeast,” 93 Proc. Natl. Acad. Sci. 5624–5628 (May 1996)) that distinctively emerges on the root of the arabidopsis thaliana, and the gene for the trivalent reduction enzyme of the arabidopsis thaliana. (Robinson et al., “The froh gene family from Arabidopsis thaliana: Putative iron-chelate reductases”, 196 Plant and Soil 245–248, Kluwer Academic Publishers (1997)).
Strategy II is an iron obtaining system that is only observed in gramineae, which is one of the monocotyledons. The gramineae emits mugineic acids that have trivalent iron chelate activity under iron deficient conditions, and absorbs iron from the root as an “Fe (III)-mugineic acid” complex. (Takagi et al., “Physiological aspect of magineic acid, a possible phytosiderophore of graminaceous plants,” 7(1–5) Journal of Plant Nutrition 469–477 (1984)). There are 7 mugineic acids (MAs) that are known: mugineic acid (MA), 2′-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA), 3-epihydroxymugineic acid (epiHMA), avenin acid (AVA), distichon acid and epihydroxydeoxymugineic acid (epiHDMA). All of the mugineic acids (MAs) are, as shown in FIG. 1, synthesized with methionine as a precursor. (Shojima et al., “Biosynthesis of Phytosiderophores”, 93 Plant Physiol. 1497–1503 (1990) and Ma et al., “Biosynthesis of Phytosiderophores in several Triticeae species with different genomes,” Vol. 50, No. 334, pp. 723–726, Journal of Experimental Botany, (1999)).
The excretion of mugineic acid has a circadian rhythm (Takagi et al. supra) and its excretion reaches a maximum after sunrise, and there is no excretion during the night. In addition, it has been observed that the granule expands before the excretion in iron deficient barley root and wilts after the excretion (Nishizawa et al., “The particular vesicle appearing in barley root cells and its relation to mugineic acid secretion,” 10(9–16) Journal of Plant Nutrition 1013–1020 (1987)). Therefore, it is believed that the mugineic acid is synthesized in this granule. These fact indicates that the responding of the gramineae to the iron deficiency is formed by not only the synthesis of the mugineic acid but also is formed by a complicated system such as the transmission of an iron deficiency signal and changes in the root form.
It has been reported that a gene for the nicotianamine synthesizing enzyme, which is an enzyme related to the mugineic acid synthesizing route, has been isolated and it is induced by an iron deficiency. (Higuchi et al., “Cloning of Nicotianamine Synthase Gene, Novel Genes Involved in the Biosynthesis of Phytosiderophore,” 119 Plant Physiology 471–479 (February 1999)). In addition, the gene for nicotianamine amino transferase (NAAT) has been isolated and it is induced by an iron deficiency. (Takahashi et al., “Purification, characterization and DNA sequencing of nicotianamine aminotransferase (NAAT-III) expressed in Fe-deficient barley roots,” Plant nutrition, 279–280, Kluwer Academic Publishers (1997))
Moreover, through differential screening using mRNA extracted from an iron deficient barley root and a control barley root, genes Ids1, Ids2, and Ids3 which were specifically induced under iron deficient conditions have been isolated. The Ids1 is a gene that codes for metallothionain protein. (Okumura et al., “An iron deficiency-specific cDNA from barley roots having two homologous cysteine-rich MT domains,” 17 Plant Molecular Biology 531–533, Kluwer Academic Publishers (1991)). Ids2 is a gene in which the sequence of amino acids that is assumed from its genetic sequence is homologous to the hydroxide enzyme. (Okumura et al., “A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare”, Plant Molecular Biology 25; 705–719, Kluwer Academic Publishers, (1994)) Ids3 is also a gene in which the sequence of amino acids that is assumed from its genetic sequence is homologous to the hydroxide enzyme. (Nakanishi et al., “Expression of a Gene Specific for Iron Deficiency (Ids3) in the Roots of Hordeum Vulgare”, 34(3) Plant Cell Physiol 401–410, JSPP (1993)) There are two hydroxide reactions along the epihydroxymugineic acid synthesizing route, and this gene is believed to code the enzyme that catalyzes this reaction.
In addition, the examples of proteins that are induced by an iron deficiency of the barley root are, the IDS3 protein, adenin-ribose-phosphate transferases (Itai et al., “Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficient barley roots: a possible role for phytosiderophore production”. Vol. 51, No. 348, pp. 1179–1188, Journal of Experimental Botany (July 2000)), formicacid dehydrogenate enzyme (Suzuki et al., “Form ate Dehydrogenase, an Enzyme of Anaerobic Metabolism, is induced by Iron Deficiency in Barley Roots,” 116 Plant Physiol 725–732 (1998)), and 36 kDa protein (Tomohiro Irifune, “Partial amino acid sequences of a specific protein in iron-deficient barley roots”, (1991)). Gramineae biosynthesizes mugineic acid under iron deficient conditions. This time, it is believed that the methionine contained in the root is reduced so that methionine is synthesized during a methionine cycle and at the same time, in order to convert the generated adenine into AMP, adenine-ribose-phosphate transferases are induced. (Itai et al., supra)
The formic acid dehydrogenate enzyme decomposes formic acid generated during the methionine cycle. It was reported that the root of a gramineae with an iron deficiency has a deformation of the mitochondrion and a reduction of the energy charge of the electron transmission system (Mori et al., “Why are young rice plants highly susceptible to iron deficiency”, Iron nutrition and interactions in plants, 175–188, Kluwer Academic Publishers (1991)). It is believed that the formic acid dehydrogenate enzyme is induced by the anaerobic condition generated by the iron deficiency, and that NADH is supplied as an energy source.
Along with the increase in population, an increase in food production is a significant issue as a condition for human existence in the future. Gramineae has been one of the most important foods since ancient times, however, in reality, the growth of gramineae is difficult in areas with iron deficiencies. If it is possible to grow graminae in an area with an iron deficiency, an increase in food production would be possible, thus it has been attracting people's attention as one of the solutions to increase food production.